FAP-activated proteasome inhibitors for treating solid tumors

ABSTRACT

Disclosed are proteasome inhibitors, fibroblast activation protein (FAP)-activated prodrugs of proteasome inhibitors, and pharmaceutically acceptable salts of the inhibitors and prodrugs. Also disclosed are related pharmaceutical compositions, and methods of using the inhibitors and prodrugs and compositions thereof, for example, in treating cancer or other cell proliferative diseases. In vitro and in vivo methods of quantifying the expression of FAP in a biopsy sample and a mammal, respectively, are also disclosed.

RELATED APPLICATIONS

This application is the U.S. national phase of International PatentApplication No. PCT/US2012/053140, filed Aug. 30, 2012, which claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.61/528,824, filed Aug. 30, 2011, the entirety of each of which isincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant CA156930awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

One in four deaths in the USA is due to cancer, the second leading causeof death after heart disease. Lung cancer is the leading cause ofmortality among cancers, and the majority of patients have locallyadvanced or metastatic non-small cell lung cancer (NSCLC) at the time ofdiagnosis. In women, breast cancer is the most prevalent cancer and isthe second leading cause of cancer-related death.

The current standard of care for treatment of solid cancers has limitedefficacy. For instance, in NSCLC survival remains poor despiteimprovements achieved by addition of targeted agents to first-lineplatinum-based chemotherapy. In metastatic breast cancer the efficacy oftrastuzumab is limited by tumor resistance. When NSCLC progresses afterfirst-line therapy, approved second-line agents only achieve modestsurvival rates.

More effective anticancer agents are clearly needed. Many approvedcancer drugs, such as bortezomib (Velcade®), are cytotoxic agents thatkill normal cells as well as tumor cells. The therapeutic benefit ofthese drugs depends on tumor cells being more sensitive than normalcells, thereby allowing clinical responses to be achieved at relativelysafe drug doses; however, damage to normal tissues is unavoidable andoften limits treatment. Following the success of bortezomib in treatingmultiple myeloma (MM), inhibition of the proteasome complex emerged as apromising new approach to chemotherapy. Due to its remarkable efficacyin treating multiple myeloma, bortezomib has been tested in solidcancers; unfortunately, it has generally failed to produce clinicalresponses.

Bortezomib inhibits an intracellular protein complex called theproteasome. The proteasome is an attractive drug target because it isinvolved in regulation of the cell cycle and apoptosis, processes thatwhen dysregulated in cancer cells lead to tumor progression, drugresistance and altered immune surveillance. By inhibiting the 20Sproteasome, which selectively degrades proteins involved in cellularhomeostasis, bortezomib stabilizes proapoptotic members of the Bcl-2family, inhibits two major pathways leading to NF-κB activation, andcauses intracellular accumulation of misfolded proteins; all of whicheffects contribute to killing tumor cells. Blockade of NF-κB activationincreases apoptosis, reduces production of angiogenic cytokines,inhibits tumor cell adhesion to stroma, and alleviates immunesuppression.

However, broader use of bortezomib to treat cancer appears to beprevented by systemic toxicity. Bortezomib distributes to healthytissues, causing diarrhea, fatigue, fluid retention, hypokalemia,hyponatremia, hypotension, malaise, nausea, orthostasis,bortezomib-induced peripheral neuropathy (BIPN) and hematologictoxicities, of which thrombocytopenia is the most severe. At therecommended dose of bortezomib there is a therapeutic window for thetreatment of MM that may be afforded by the unique sensitivity of MMcells to inhibition of nuclear factor-κB (NF-κB) and induction of theunfolded protein response. Solid cancers (e.g., prostate, pancreatic andbreast cancer) appear to be less sensitive, however, and attempts toachieve efficacy by increasing bortezomib dosage have been prevented bydose-limiting toxicities (DLTs). The poor localization of bortezomib totumors appears to contribute to its low therapeutic index (TI) in solidcancers. In mice bearing PC3 prostate tumors, healthy organ exposure to¹⁴C-bortezomib was as much as 9-fold greater than tumor exposure, andproteasome inhibition in healthy tissue appears to be greater than insolid tumors. Thus, it is necessary to design compounds that selectivelytarget the proteasome in tumor cells to overcome the obstacle of DLTsdue to proteasome inhibition in healthy tissues.

Extensive efforts over the past few decades have focused on therapiestailored to the specific patient—so-called personalized medicine. Due toadvances in genetic sequencing technology it is now possible andincreasingly cost-effective to genotype cancerous tissue to identify theindividual genetic profile of the cancer and thus the specific mutatedor dysfunctional proteins that may be responsible for tumor growth. Such“driver” proteins may be then targeted with agents that block theirfunction and thus kill the cancer. While conceptually sound, thisapproach has been hampered by the unexpected genetic diversity andgenomic instability of cancer. Significantly different genotypes ofcancer may be present within a single tumor, making targeted therapyineffective for many patients. Even when the majority of cancer cells ina tumor share a sufficiently similar genetic makeup that a singletargeted therapy is effective, small numbers of cancer cells bearing aresistant mutation may survive the therapy, leading to relapse after aninitial improvement.

Therapies selectively targeting the tumor and its microenvironment withcytotoxic agents whose effect does not depend on the genetic makeup ofthe cancer are needed. Such therapies remain elusive, however.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a FAP-activated prodrugof a proteasome inhibitor represented by A-B, or a pharmaceuticallyacceptable salt thereof, wherein

-   -   A represents a substrate for Fibroblast Activation Protein        (FAP);    -   B represents a proteasome inhibitor moiety which, when released        in a free form from the prodrug as a product of cleavage by FAP,        inhibits the proteolytic activity of a proteasome with a Ki of        500 nM or less;    -   A and B being covalently linked by a bond that is enzymatically        cleaved by FAP to release B in said free form; and    -   the prodrug has a k_(cat)/K_(m) for FAP cleavage of the bond        linking A and B of at least 10 fold more than for prolyl        endopeptidase EC 3.4.21.26 (PREP).    -   Another aspect of the present invention relates to a        FAP-activated proteasome inhibitor represented by formula I:

or a pharmaceutically acceptable salt thereof,

-   -   wherein    -   X—C(═O)NR₁₁—R′₅— represents the FAP substrate sequence, X is an        N-acyl peptidyl group, —NR₁₁—R′₅ is an amino acid residue or        analog thereof that binds the P′₁ specificity subsite of FAP,        and the FAP substrate sequence is cleaved by FAP to release        NHR₁₁—R′₅—R; and        -   NHR₁₁—R′₅—R is a proteasome inhibitor.

Another aspect of the present invention relates to a compound or apharmaceutically acceptable salt thereof represented by the formula:R-Xaa₁-Xaa₂-YwhereinR is an acyl group;Xaa₁ is selected from the group consisting of Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp,and Tyr;Xaa₂ is selected from the group consisting of Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp,and Tyr; andY is

Another aspect of the present invention relates to pharmaceuticalcompositions, and methods of using the compounds and compositions in,for example, treating cancer or other cell proliferative diseases.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the synthesis of ARI-2727D and ARI-3996. The followingreagents were used: (a) HATU/DMF/DIPEA, 95%; (b) 4 M HCl in dioxane,100%; (c) HATU/DMF/DIPEA, 90%; (d) 4M HCl in dioxane, 100%; (e)tBu-Suc-Ser(tBu)-OH, HATU/DMF/DIPEA, 90%; (f) Pd(OH)₂—C/H₂/methanol,90%; (g) 2727D, HATU/DMF/DIPEA, 85%; (h) TFA/DCM, 90%; (i) PhB(OH)₂,pentane-water-acetonitrile, 70%.

FIG. 2 shows the rate of in vitro cleavage of ARI-3996 by FAP and PREPas a function of the concentration of ARI-3996. Cleavage was monitoredby assay of release of “warhead” ARI-2727 using LCMS.

FIG. 3 shows in vitro cleavage of ARI-3144((N-Quinoline-4-carbonyl)-D-Ala-Pro-AMC) by FAP, but not by DPP IV, DPP8, DPP 9 or PREP. Cleavage was monitored by measuring fluorescence ofthe AMC leaving group (excitation, 355 nm; emission, 460 nm).

FIG. 4 shows the ratio of FAP proteolytic activity in humans and micefor pancreatic tumor tissue and plasma. FAP activity was assayed ex vivoin tumor homogenates and plasma using ARI-3144 fluorogenic substrate.

FIG. 5 shows FAP proteolytic activity in xenografts of FAP-transfectedHEK293 cells and HPAF-11 cells. FAP activity was assayed using theARI-3144 assay.

FIG. 6 shows a comparison of antitumor activities of ARI-3996 andbortezomib (Velcade®) at respective MTDs in SCID mice bearingestablished (˜200 mm³) HPAF-II carcinoma xenografts. ARI-3996 andbortezomib were administered twice weekly (day (D)1/D4 schedule), andARI-3996 was also given from day 1 to day 5 for 5 consecutive days (QDx5schedule). Asterisks indicate significant (p<0.05) reductions in tumorsize in mice treated with ARI-3996, compared to controls.

FIG. 7 shows the distribution of [¹⁴C] bortezomib to tissues and tumor 1hour after i.v. injection. The graph and data were taken from Adams etal (71). Data are mean dpm/100 mg tissue and mean dpm/100 μL blood.

FIG. 8 shows tissue distribution of ARI-3996 and the warhead 2727D inSCID mice bearing HPAF-II s.c. tumors. Tumor-bearing mice were injecteds.c. with 50 mg/kg ARI-3996. Tissues were harvested at 1 (A, B), 2 (C,D) and 3 hours (n=2) after administration of ARI-3996, and drugconcentrations in tissue extracts were determined by LCMS.

FIG. 9 shows the antitumor effect of ARI-3996 administered by i.p. (IP)and s.c. (SQ) routes at 50 mg/kg twice daily (b.i.d) on days 1 and 4 toSCID mice bearing HPAF-II xenografts. One-way ANOVA with Dunnett's posttest for test versus vehicle (P<0.0001).

FIG. 10 shows antitumor activity of 50 mg/kg ARI-3996 with or without 6mg/kg gemcitabine. ARI-3996 b.i.d. s.c. and gemcitabine once per dayi.p. were administered twice weekly. Mean±SEM. The two compounds exhibita strong synergistic effect when administered together.

FIG. 11 shows a cartoon of diagnostic fluorogenic substrate ARI-3144.The FAP recognition site binds specifically to FAP and is cleaved by theenzyme to release the fluorogenic coumarin moiety.

FIG. 12 shows a cartoon of ARI-3144 after binding to FAP. The FAPrecognition site is cleaved to release the fluorescent coumarin moiety.

FIG. 13 shows that ARI-3144 is an excellent substrate for FAP.

FIG. 14 shows fluorescence measurements of the rate of ARI-3144 cleavageby PREP, DPPIV, DPP8, DPP9, and DPPII. ARI-3144 is highly selective forFAP.

FIG. 15 shows a cartoon of the prodrug ARI-3996, which contains a FAPrecognition site chemically bound to ARI-2727D, a proteasome inhibitorwhich remains inactive while bound to the FAP recognition site (top) anda cartoon of what takes place after cleavage by FAP; the active“warhead” ARI-2727D is released from the FAP recognition site (bottom).

FIG. 16 shows FAP activity in cancerous and normal mouse tissues. Themuch higher FAP activity in and around the tumor indicates that FAP isupregulated in that tissue.

FIG. 17 shows FAP activity in human tumor cell lines and HPAF-II mousetumor xenographs. FAP activity is generally higher in human tumor celllines than mouse tumor xenographs. FAP activity is likely to be evenhigher than shown due to some deactivation of FAP during samplecollection and handling.

FIG. 18 shows a graph of FAP activity in several tissues. FAPtransfected HEK tumor xenographs match human pancreatic tumor tissue forFAP content.

FIG. 19 shows the mean tumor volume of mice treated with either avehicle control or with ARI-3996. ARI-3996 induces tumor regression inimmunocompetent mice.

FIG. 20 shows the anticancer activity of ARI-3996 in FAP-transfected HEKtumor xenograft.

FIG. 21 shows that FAP knockout mouse blood plasma does not activateARI-3996 to release ARI-2727.

FIG. 22 shows tissue distribution of Velcade® versus ARI-3996 in mice.

FIG. 23 shows tissue distribution of Velcade® versus ARI-2727D in mice.ARI-3996 is cleaved to ARI-2727D in and around tumors, thus facilitatingthe buildup of ARI-2727D in tumors.

FIG. 24 shows the tissue distribution of ARI-2727D 1 hour after directadministration vs. administration as the prodrug form, ARI-3996 (top);and the average ratio at which ARI-2727D accumulates in the tumor versusthe liver.

FIG. 25 shows the cytotoxicity of Velcade® versus ARI-2727D in multiplemyeloma, normal cells, and solid tumors.

FIG. 26 shows the cytotoxicity of Velcade® versus ARI-2727D versusARI-3996 in multiple myeloma, normal cells, and solid tumors.

FIG. 27 shows the FAP activity in various human cancer cell lines.

FIG. 28 shows the anticancer effects of proteasome inhibitors Velcade®and ARI-3996 in U266 tumor-bearing mice.

FIG. 29 shows the chemical structures and names of a number of knownproteasome inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds designed selectively totarget solid tumors with a reduced toxicity profile. Bortezomib(Velcade®) is an effective treatment for multiple myeloma, but itsmechanism of action results in dose-limiting toxicities (DLTs) ofperipheral neuropathy and loss of platelets, which prevent treatment ofcommon solid cancers. The compounds of the present invention aredesigned to remain inactive in healthy organs and to be activated by thetumor-associated enzyme called fibroblast activation protein (FAP) tounleash a cytotoxic bortezomib-like warhead in tumors, thereby reducingthe toxic side effects that prevent safe treatment of solid tumors withbortezomib.

The selective targeting and reduced toxicity of the compounds of theinvention allows the treatment of solid cancers independent of theirgenetic makeup. Furthermore, the selective activation of the compoundsin the vicinity of the tumors results in a high concentration of thecytotoxic agent in the tumor but a low concentration in the rest of thebody. The high local concentration kills tumors with a lower dose of thedrug than previously possible, because a drug lacking the capability tobe selectively delivered circulates throughout the body, causingsystemic toxicity, often at a dose that is suboptimal for treatment ofthe cancer.

The present invention also allows the offsetting of theimmunosuppressive properties of tumors. Because solid tumors are oftensurrounded by cancerous stromal cells, they are protected from theimmune system of the patient. This immunosuppression can be removed bykilling the stromal cells, but conventional chemotherapies includingVelcade® fail to do so. The present invention is capable of killingstromal cells because they overexpress FAP and thus activate thecompounds of the invention to release the warhead. Thus the presentinvention can have multiple mechanisms of action, such as direct killingof tumors or re-activation of the patient immune response after killingof the supportive stromal tissue, resulting in killing of the tumorthrough a natural immune response.

The FAP address moiety, or FAP binding portion, of the invention may bechemically attached to a variety of cytotoxic warheads. Thus, anyproteasome inhibitor with a validated target and mode of action wouldbenefit from use with the claimed invention. Conjugation (chemicalattachment) of a validated proteasome inhibitor possessing anticanceractivity, to the FAP address moiety confers selective delivery,increased potency, and decreased off-target toxicity.

Conjugation of the FAP address moiety to a known protease inhibitor issimilar to, but conceptually different from, a prodrug, because the FAPaddress moiety is designed to bind and be cleaved by FAP selectivelyover other proteases present in the body, especially DPPII, DPP8, DPP9,DPPIV, and PREP. This specificity for enzyme subtype is essential forthe desired effect of delivering the released cytotoxic agent to thetumor.

Many proteasome inhibitors with anticancer activity are known in theart, and may be divided according to covalent and non-covalentinhibitors, with the covalent inhibitors further divided into aldehydes,boronates, epoxyketones, beta-lactones, vinyl sulfones, andα,β-unsaturated carbonyls, among others. Examples in the aldehyde classinclude MG-132, PSI, and fellutamide B. Examples in the boronate classinclude bortezomib (Velcade®), CEP-18770, MLN2238, and MLN9708. Examplesin the epoxyketone class include epoxomicin, carfilzomib (PR-171),NC-005, YU-101, LU-005, YU-102, NC-001, LU-001, NC-022, PR-957 (LMP7),CPSI (β5), LMP2-sp-ek, BODIPY-NC-001, azido-NC-002, and ONX 0912(opromozib). Examples in the beta-lactone class include omuralide,PS-519, marizomib, and belactosin A. Examples in the vinyl sulfone classinclude ¹²⁵I-NIP-L₃VS, NC-005-VS, and MV151. Discussion and validationof these inhibitors and others may be found, for example, in Kisselev etal. “Proteasome Inhibitors: An Expanding Army Attacking a UniqueTarget,” Chemistry and Biology 19, Jan. 27, 2012, 99-115 (incorporatedby reference).

Chemical conjugation of any of these proteasome inhibitors with a FAPaddress moiety as described in the present invention would be expectedto deliver selectively the cytotoxic agent to solid tumors and thesurrounding stromal cells. Since the FAP address moiety is a selectivesubstrate for FAP, the identity of the cytotoxic agent attached to theFAP address moiety is not important to the selective delivery. FAP willcleave the chemical bond attaching the address moiety to the warhead;such a chemical bond may be, for example, an ester or amide bond, amongothers.

One aspect of the present invention relates to a FAP-activated prodrugof a proteasome inhibitor represented by A-B, or a pharmaceuticallyacceptable salt thereof, wherein

-   -   A represents a substrate for Fibroblast Activation Protein        (FAP);    -   B represents a proteasome inhibitor moiety which, when released        in a free form from the prodrug as a product of cleavage by FAP,        inhibits the proteolytic activity of a proteasome with a Ki of        500 nM or less;    -   A and B being covalently linked by a bond that is enzymatically        cleaved by FAP to release B in said free form; and    -   the prodrug has a k_(cat)/K_(m) for FAP cleavage of the bond        linking A and B of at least 10 fold more than for prolyl        endopeptidase EC 3.4.21.26 (PREP).

In certain embodiments, the free form of said proteasome inhibitormoiety has an IC₅₀ for inhibiting proteasome activity of cells in vitrothat is at least 10 fold less relative to said prodrug.

In certain embodiments, the free form of said proteasome inhibitormoiety has a K_(i) for inhibiting proteasome activity that is at least10 fold less relative to said prodrug.

In certain embodiments, the free form of said proteasome inhibitormoiety has at least 5 fold greater cell permeability into human cellsthan said prodrug.

In certain embodiments, the prodrug has a therapeutic index in vivo atleast 5 fold greater than said free form of said proteasome inhibitormoiety.

In certain embodiments, the prodrug has a therapeutic index in vivo ofat least 10.

In certain embodiments, the prodrug has a maximum tolerated dose atleast 10 times greater than[(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronicacid.

In certain embodiments, said free form of said proteasome inhibitormoiety is a dipeptidyl moiety, which when released from the prodrug asan open chain product of cleavage by FAP, undergoescyclization-dependent inactivation over time.

In certain embodiments, said open chain product undergoescyclization-dependent inactivation with a T_(1/2) of 5 hours or less.

In certain embodiments, A represents a peptide or peptide analogue whichis a substrate for FAP, which peptide or peptide analogue includes anN-terminal blocking group.

In certain embodiments, the peptide or peptide analogue is 2-10 aminoacid residues in length.

In certain embodiments, the peptide or peptide analogue is C-terminallylinked to B.

In certain embodiments, at least one amino acid residue of the peptideor peptide analog is a non-naturally occurring amino acid analog.

In certain embodiments, the N-terminal blocking group is a moiety which,at physiological pH, reduces the cell permeability of said prodrugrelative to said free form of said proteasome inhibitor.

In certain embodiments, the N-terminal blocking group includes one ormore functional groups that are ionized at physiological pH.

In other embodiments, the N-terminal blocking group is a (loweralkyl)-C(═O)-substituted with one or more functional groups that areionized at physiological pH.

In certain other embodiments, the N-terminal blocking group isrepresented by the formula —C(═O)—(CH₂)₁₋₁₀—C(═O)—OH.

In certain embodiments, the N-terminal blocking group includes one ormore carboxyl groups. In another embodiment, the N-terminal blockinggroup is succinyl.

In certain embodiments, B is a covalent or non-covalent proteasomeinhibitor.

In certain other embodiments, B is a covalent proteasome inhibitor.

In certain embodiments, B is a dipeptidyl moiety having at its carboxyterminus an electrophilic functional group that can form a covalentadduct with an amino acid residue in the active site of a proteasome.

In certain embodiments, the electrophilic functional group is analdehyde, boronic acid, boronate ester, epoxyketone, beta-lactone, vinylsulfone, or α,β-unsaturated carbonyl.

In certain embodiments, the electrophilic functional group is analdehyde, boronic acid, or epoxyketone.

In another embodiment, the electrophilic functional group is anepoxyketone.

In certain embodiments, B is selected from the group consisting of:

In certain embodiments, B is selected from the group consisting of:

In certain other embodiments, B is selected from the group consistingof:

Another aspect of the present invention relates to the compoundsdescribed above, wherein A further comprises a self-eliminating linkerattached to B by a chemical bond.

In certain embodiments, the self-eliminating linker isp-aminobenzyloxocarbonyl (PABC) or 2,4-bis(hydroxymethyl)aniline.

Another aspect of the present invention relates to a compound or apharmaceutically acceptable salt thereof represented by the formula:R-Xaa₁-Xaa₂-YwhereinR is an acyl group;Xaa₁ is selected from the group consisting of Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp,and Tyr;Xaa₂ is selected from the group consisting of Ala, Cys, Asp, Glu, Phe,Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp,and Tyr; andY is

In certain embodiments, the compound further comprises aself-eliminating linker with a chemical bond to the carboxyl terminus ofXaa₂ and a chemical bond to Y.

In certain embodiments, the self-eliminating linker isp-aminobenzyloxocarbonyl (PABC) or 2,4-bis(hydroxymethyl)aniline.

In certain embodiments, R is selected from the group consisting offormyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl.

In certain other embodiments, R is succinyl or methoxysuccinyl.

In another embodiment, R is succinyl.

In certain embodiments, Xaa₁ is Cys, Met, Ser, or Thr.

In certain other embodiments, Xaa₁ is Ser.

In certain embodiments, Xaa₂ is Ala, Gly, Ile, Leu, or Val.

In certain embodiments, Xaa₂ is Ala.

In certain other embodiments, Xaa₂ is (D)-Ala.

Y is

In certain embodiments, R is selected from the group consisting offormyl, acetyl, benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl;Xaa₁ is Cys, Met, Ser, or Thr; Xaa₂

is Ala, Gly, Ile, Leu, or Val; and Y is

In certain embodiments, R is succinyl or methoxysuccinyl; Xaa₁ is Ser;Xaa₂ is Ala;

and Y is

In certain embodiments, R is succinyl; Xaa₁ is Ser; Xaa₂ is (D)-Ala; andY is

In certain embodiments, the present invention relates to a compound or apharmaceutically acceptable salt thereof represented by

In certain embodiments, the compound is represented by

Another aspect of the present invention relates to a FAP-activatedproteasome inhibitor represented by formula I:

or a pharmaceutically acceptable salt thereof,

wherein

-   -   X—C(═O)NR₁—R′₅— represents the FAP substrate sequence, X is an        N-acyl peptidyl group, —NR₁₁—R′₅ is an amino acid residue or        analog thereof that binds the P′₁ specificity subsite of FAP,        and the FAP substrate sequence is cleaved by FAP to release        NHR₁₁—R′₅—R; R₁₁ represents H or lower alkyl; and        -   NHR₁₁—R′₅—R is a proteasome inhibitor.    -   In certain embodiments, the present invention relates to the        FAP-activated proteasome inhibitor described above, represented        by formula II:

wherein

-   -   R₁—(C═O)— represents an acyl N-terminal blocking group;    -   R₂ represents H, lower alkyl, or a mono- or        di-hydroxy-substituted lower alkyl;    -   R₃ represents H, halogen, or lower alkyl;    -   R₄ is absent or represents lower alkyl, —OH, —NH₂ or halogen;    -   R₅ represents a large hydrophobic amino acid sidechain;    -   R₁₁ represents H or lower alkyl; and        -   the FAP-activated proteasome inhibitor is cleaved by FAP to            release a proteasome inhibitor represented by

In certain embodiments of the FAP-activated proteasome inhibitor,

is selected from the group consisting of:

In certain embodiments, the FAP-activated proteasome inhibitor isrepresented by formula III:

wherein

R₁—(C═O)— represents an acyl N-terminal blocking group;

R₂ represents H, lower alkyl, or a mono- or di-hydroxy-substituted loweralkyl;

R₃ represents H, halogen, or lower alkyl;

R₄ is absent or represents lower alkyl, —OH, —NH₂ or halogen;

R₅ represents a large hydrophobic amino acid sidechain;

R₆ represents alkyl, cycloalkyl, aryl, heterocycle or —(CH₂)_(n)—R₇;

R₇ represents aryl, aralkyl, cycloalkyl, alkoxy, alkylthio, —OH or —SH;

R₁₁ represents H or lower alkyl;

W represents —CN, an epoxyketone, —CH═NR₅,

R₈ represents H, alkyl, alkenyl, alkynyl, —C(X₁)(X₂)X₃, —(CH₂)_(m)—R₉,—(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl, —(CH₂)_(n)—O-alkenyl,—(CH₂)_(n)—O-alkynyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₉, —(CH₂)_(n)—SH,—(CH₂)_(n)—S-alkyl, —(CH₂)_(n)—S-alkenyl, —(CH₂)_(n)—S-alkynyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₉, —C(═O)C(═O)NH₂, —C(═O)C(═O)OR₁₀;

R₉ represents, independently for each occurrence, a substituted orunsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R₁₀ represents, independently for each occurrence, hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle;

Y₁ and Y₂ can independently or together be OH, or a group capable ofbeing hydrolyzed to a hydroxyl group, including cyclic derivatives whereY₁ and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure;

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₁₀;

R₅₂ represents hydrogen, lower alkyl, amine, —OR₁₀, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure;

X₁ is halogen;

X₂ and X₃ each represent H or halogen;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

-   -   In certain embodiments, the present invention relates to the        FAP-activated proteasome inhibitor described above, represented        by

Another aspect of the present invention relates to a pharmaceuticalcomposition, comprising a compound described herein; and apharmaceutically acceptable excipient.

Another aspect of the present invention relates to a method ofinhibiting proteasome function in a cell, comprising contacting the cellwith an effective amount of a compound described herein.

Another aspect of the present invention relates to a method of reducingthe rate of muscle protein degradation in a cell, comprising contactingthe cell with an effective amount of a compound described herein.

Another aspect of the present invention relates to a method of reducingthe activity of NF-κB in a cell, comprising contacting the cell with aneffective amount of a compound described herein.

Another aspect of the present invention relates to a method of reducingthe rate of proteasome-dependent intracellular protein breakdown,comprising contacting a cell with an effective amount of a compounddescribed herein.

Another aspect of the present invention relates to a method of reducingthe rate of degradation of p53 protein in a cell, comprising contactingthe cell with an effective amount of a compound described herein.

Another aspect of the present invention relates to a method ofinhibiting cyclin degradation in a cell, comprising contacting the cellwith an effective amount of a compound described herein.

Another aspect of the present invention relates to a method ofinhibiting antigen presentation in a cell, comprising contacting thecell with an effective amount of a compound described herein.

Another aspect of the present invention relates to a method of treatingcancer, psoriasis, restenosis, or other cell proliferative disease,comprising administering to a mammal in need thereof a therapeuticallyeffective amount of a compound described herein.

Another aspect of the present invention relates to a method of treatingcancer, psoriasis, restenosis, or other cell proliferative disease,comprising co-administering to a mammal in need thereof atherapeutically effective amount of a compound described herein; and atherapeutically effective amount of a chemotherapeutic agent.

In certain embodiments, the chemotherapeutic agent is docetaxel,paclitaxel, imatinib mesylate, gemcitabine, cis-platin, carboplatin,5-fluorouracil, pemetrexed, methotrexate, doxorubicin, lenalidomide,dexamethasone, or monomethyl auristatin.

In certain other embodiments, the chemotherapeutic agent is docetaxel,gemcitabine, carboplatin, or doxorubicin.

In yet other embodiments, the chemotherapeutic agent is MG-132, PSI,fellutamide B, bortezomib, CEP-18770, MLN-2238, MLN-9708, epoxomicin,carfilzomib (PR-171), NC-005, YU-101, LU-005, YU-102, NC-001, LU-001,NC-022, PR-957 (LMP7), CPSI (β5), LMP2-sp-ek, BODIPY-NC-001,azido-NC-002, ONX-0912, omuralide, PS-519, marizomib, belactosin A,¹²⁵I-NIP-L₃VS, NC-005-VS, or MV151.

Another aspect of the present invention relates to a method of treatingcancer, comprising administering to a mammal in need thereof atherapeutically effective amount of a compound described herein.

In certain embodiments, the cancer is a solid tumor.

In certain other embodiments, the method further comprises administeringto a mammal in need thereof a therapeutically effective amount of achemotherapeutic agent.

In yet other embodiments, the cancer is a solid tumor.

In still yet other embodiments, the chemotherapeutic agent is docetaxel,paclitaxel, imatinib mesylate, gemcitabine, cis-platin, carboplatin,5-fluorouracil, pemetrexed, methotrexate, doxorubicin, lenalidomide,dexamethasone, or monomethyl auristatin.

In another embodiment, the chemotherapeutic agent is docetaxel,gemcitabine, carboplatin, or doxorubicin.

In certain embodiments, the chemotherapeutic agent is MG-132, PSI,fellutamide B, bortezomib, CEP-18770, MLN-2238, MLN-9708, epoxomicin,carfilzomib (PR-171), NC-005, YU-101, LU-005, YU-102, NC-001, LU-001,NC-022, PR-957 (LMP7), CPSI (β5), LMP2-sp-ek, BODIPY-NC-001,azido-NC-002, ONX-0912, omuralide, PS-519, marizomib, belactosin A,¹²⁵I-NIP-L₃VS, NC-005-VS, or MV151.

Another aspect of the present invention relates to method of reducingthe rate of loss of muscle mass in a mammal, comprising administering toa mammal in need thereof a therapeutically effective amount of acompound described herein.

Another aspect of the present invention relates to a method of reducingthe activity of NF-κB in a mammal, comprising administering to a mammalin need thereof a therapeutically effective amount of a compounddescribed herein.

Another aspect of the present invention relates to a method of reducingthe rate of proteasome-dependent intracellular protein breakdown in amammal, comprising administering to a mammal in need thereof atherapeutically effective amount of a compound described herein.

Another aspect of the present invention relates to a method of reducingthe rate of degradation of p53 protein in a mammal, comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of a compound described herein.

Another aspect of the present invention relates to a method ofinhibiting cyclin degradation in a mammal, comprising administering to amammal in need thereof a therapeutically effective amount of a compounddescribed herein.

Another aspect of the present invention relates to a method ofinhibiting antigen presentation in a mammal, comprising administering toa mammal in need thereof a therapeutically effective amount of acompound described herein.

Another aspect of the present invention relates to a method ofinhibiting inducible NF-κB dependent cell adhesion in a mammal,comprising administering to a mammal in need thereof a therapeuticallyeffective amount of a compound described herein.

Another aspect of the present invention relates to a method ofinhibiting HIV infection in a mammal, comprising administering to amammal in need thereof a therapeutically effective amount of a compounddescribed herein.

Another aspect of the present invention relates to a method ofquantifying the amount of FAP expressed by or in the vicinity of a tumorin a mammal, comprising the steps of:

administering to said mammal an effective amount of a compoundrepresented by Formula IV:

-   -   wherein R₁₂ is a fluorophore or chromophore;

illuminating the mammal in the vicinity of the tumor; and

measuring the amount of fluorescence in the vicinity of the tumor.

In certain embodiments, R₁₂ is selected from the group consisting of:

In certain other embodiments, R₁₂ is

Another aspect of the present invention relates to a method ofquantifying the amount of FAP expressed by a tumor biopsy sample,comprising the steps of:

combining said tumor biopsy sample with an effective amount of acompound represented by Formula IV, thereby forming a mixture:

-   -   wherein R₁₂ is a fluorophore or chromophore;

illuminating the mixture; and

measuring the amount of fluorescence in the mixture.

In certain embodiments, R₁₂ is selected from the group consisting of:

In certain other embodiments, R₁₂ is

Another aspect of the present invention relates to a method describedherein, wherein said mammal is a primate, equine, canine, feline, orbovine.

Another aspect of the present invention relates to a method describedherein, wherein said mammal is a human.

Another aspect of the present invention relates to a method describedherein, wherein the compound is administered to the mammal byinhalation, orally, intravenously, sublingually, ocularly,transdermally, rectally, vaginally, topically, intramuscularly,intra-arterially, intrathecally, subcutaneously, buccally, orintranasally.

Another aspect of the present invention relates to a method describedherein, wherein the compound is administered to the mammalintravenously.

Another aspect of the present invention relates to a method for reducinglocal immunosuppression and/or tumor supporting-activity mediated byFAP+ stromal cells, comprising administering to a patient in needthereof a therapeutically effective amount of a prodrug of an activeagent, wherein the active agent is cytotoxic or inhibits proteinexpression or secretion to said FAP+ stromal cells, and is at least 2fold more cytotoxic to the FAP+ stromal cells than the prodrug; and theprodrug (i) includes an FAP substrate sequence; (ii) is converted to theactive agent by cleavage of the FAP substrate sequence by FAP, whichsubstrate sequence has a k_(cat)/K_(m) for cleavage by FAP at least 10fold more than for cleavage by prolyl endopeptidase EC 3.4.21.26 (PREP);and (iii) is selectively converted in vivo to the active agent by FAP+stromal cells.

Another aspect of the present invention relates to a method describedherein, wherein the FAP substrate sequence has a k_(cat)/K_(m) forcleavage by FAP at least 10 fold more than for cleavage by other S9prolyl endopeptidases.

Another aspect of the present invention relates to a method describedherein, wherein the prodrug is represented by formula V:

or a pharmaceutically acceptable salt thereof,

wherein

-   -   X—C(═O)NR₁₁—R′₅— represents the FAP substrate sequence, X is an        N-acyl peptidyl group, —NR₁₁—R′₅ is an amino acid residue or        analog thereof that binds the P′₁ specificity subsite of FAP,        and the FAP substrate sequence is cleaved by FAP to release        NHR₁₁—R′₅—R; R₁₁ represents H or lower alkyl; and    -   R′₅ and R taken together form the cytotoxic agent, or a moiety        further metabolized at the site of the FAP+ stomal cells to form        the cytotoxic agent.

Another aspect of the present invention relates to a method describedherein, wherein the prodrug is represented by formula VI:

or a pharmaceutically acceptable salt thereof,

wherein

-   -   R₁—C(═O)— represents an acyl N-terminal blocking group;    -   Xaa(1) is an amino acid residue;    -   Xaa(2) is glycine, or a (D)-amino acid residue;    -   PRO represents a proline residue or an analog thereof;    -   Xaa(3) is a large hydrophobic amino acid residue; and    -   the prodrug is cleaved by FAP to release Xaa(3)-R, and Xaa(3)-R        is the cytotoxic agent.

Another aspect of the present invention relates to a method describedherein, wherein the prodrug is represented by formula VII:

or a pharmaceutically acceptable salt thereof,

wherein

-   -   R₁—(C═O)— represents an acyl N-terminal blocking group;    -   R₂ represents H, lower alkyl, or a mono- or        di-hydroxy-substituted lower alkyl;    -   R₃ represents H, halogen, or lower alkyl;    -   R₄ is absent or represents lower alkyl, —OH, —NH₂ or halogen;    -   R₅ represents a large hydrophobic amino acid sidechain;    -   R₁₁ represents H or lower alkyl; and        the prodrug is cleaved by FAP to release the cytotoxic agent,

In certain embodiments, the acyl N-terminal blocking group is a moietywhich, at physiological pH, reduces the cell permeability of saidprodrug relative to said cytotoxic agent.

In certain embodiments, the acyl N-terminal blocking group is selectedfrom the group consisting of formyl, acetyl, benzoyl, trifluoroacetyl,succinyl and methoxysuccinyl.

In certain embodiments, the acyl N-terminal blocking group includes oneor more functional groups that are ionized at physiological pH.

In certain embodiments, the acyl N-terminal blocking group includes oneor more carboxyl groups.

In certain embodiments, the acyl N-terminal blocking group is (loweralkyl)-C(═O)-substituted with one or more functional groups that areionized at physiological pH.

In certain embodiments, the acyl N-terminal blocking group is selectedfrom the group consisting of aryl(C₁-C₆)acyl, and heteroaryl(C₁-C₆)acyl.

In certain embodiments, the acyl N-terminal blocking group is anaryl(C₁-C₆)acyl, wherein aryl(C₁-C₆)acyl is a (C₁-C₆)acyl substitutedwith an aryl selected from the group consisting of benzene, naphthalene,phenanthrene, phenol and aniline.

In certain embodiments, the acyl N-terminal blocking group is anheteroaryl(C₁-C₆)acyl, wherein heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with a heteroaryl selected from the group consisting ofpyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine.

In certain embodiments, the acyl N-terminal blocking group isrepresented by the formula —C(═O)—(CH₂)₁₋₁₀—C(═O)—OH.

In certain embodiments, the acyl N-terminal blocking group is succinyl.

In certain embodiments, at least one of Xaa(1), Xaa(2) and Xaa(3) is anon-naturally occurring amino acid analog.

Another aspect of the invention relates to a method described herein,wherein the cytotoxic agent is a proteasome inhibitor.

Another aspect of the invention relates to a method described herein,wherein the proteasome inhibitor is represented by formula VIII:

or a pharmaceutically acceptable salt thereof,

wherein

-   -   R₁—(C═O)— represents an acyl N-terminal blocking group;    -   R₂ represents H, lower alkyl, or a mono- or        di-hydroxy-substituted lower alkyl;    -   R₃ represents H, halogen, or lower alkyl;    -   R₄ is absent or represents lower alkyl, —OH, —NH₂ or halogen;    -   R₅ represents a large hydrophobic amino acid sidechain;    -   R₆ is alkyl, cycloalkyl, aryl, heterocycle or —(CH₂)_(n)—R₇;        -   R₇ is aryl, aralkyl, cycloalkyl, alkoxy, alkylthio, —OH or            —SH;    -   R₁₁ represents H or lower alkyl;    -   W represents —CN, an epoxyketone, —CH═NR₅,

-   -   R₈ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,        —(CH₂)_(m)—R₉, —(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkyl,        —(CH₂)_(n)—O-alkenyl, —(CH₂)_(n)—O-alkynyl,        —(CH₂)_(n)O—(CH₂)_(m)—R₉, —(CH₂)_(n)—SH, —(CH₂)_(n)—S-alkyl,        —(CH₂)_(n)—S-alkenyl, —(CH₂)_(n)—S-alkynyl,        —(CH₂)_(n)—S—(CH₂)_(m)—R₉, —C(═O)C(═O)NH₂, —C(═O)C(═O)OR₁₀;    -   R₉ represents, independently for each occurrence, a substituted        or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or        heterocycle;    -   R₁₀ represents, independently for each occurrence, hydrogen, or        a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl,        cycloalkyl, cycloalkenyl, or heterocycle;    -   Y₁ and Y₂ can independently or together be OH, or a group        capable of being hydrolyzed to a hydroxyl group, including        cyclic derivatives where Y₁ and Y₂ are connected via a ring        having from 5 to 8 atoms in the ring structure;    -   R₅₀ represents O or S;    -   R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₁₀;    -   R₅₂ represents hydrogen, lower alkyl, amine, —OR₁₀, or a        pharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together        with the phosphorous atom to which they are attached complete a        heterocyclic ring having from 5 to 8 atoms in the ring        structure;    -   X₁ is halogen;    -   X₂ and X₃ each represent H or halogen; and    -   m is zero or an integer in the range of 1 to 8; and n is an        integer in the range of 1 to 8.

In certain embodiments, the proteasome inhibitor is

In certain embodiments, the prodrug has a therapeutic index at least twotimes greater than the therapeutic index for the proteasome inhibitorwhen administered alone.

Another aspect of the invention relates to a method described herein,wherein the prodrug is administered as a single agent therapy.

Another aspect of the invention relates to a method described herein,wherein the prodrug is administered in a combination therapy with one ormore anti-cancer agents.

Another aspect of the invention relates to a method described herein,wherein the anti-cancer agent is a covalent proteasome inhibitor.

Another aspect of the invention relates to a method described herein,wherein the anti-cancer agent is a chemotherapeutic.

Another aspect of the invention relates to a method described herein,wherein the chemotherapeutic is docetaxel, paclitaxel, imatinibmesylate, gemcitabine, cis-platin, carboplatin, 5-fluorouracil,pemetrexed, methotrexate, doxorubicin, lenalidomide, dexamethasone, ormonomethyl auristatin.

Another aspect of the invention relates to a method described herein,wherein the chemotherapeutic is docetaxel, gemcitabine, carboplatin, ordoxorubicin.

Another aspect of the invention relates to a method described herein,wherein the chemotherapeutic is MG-132, PSI, fellutamide B, bortezomib,CEP-18770, MLN-2238, MLN-9708, epoxomicin, carfilzomib (PR-171), NC-005,YU-101, LU-005, YU-102, NC-001, LU-001, NC-022, PR-957 (LMP7), CPSI(β5), LMP2-sp-ek, BODIPY-NC-001, azido-NC-002, ONX-0912, omuralide,PS-519, marizomib, belactosin A, ¹²⁵I-NIP-L₃VS, NC-005-VS, or MV151.

Another aspect of the invention relates to a method described herein,wherein the anti-cancer agent is an immunotherapeutic agent.

Another aspect of the invention relates to a method described herein,wherein the immunotherapeutic agent is an anti-tumor antibody.

Another aspect of the invention relates to a method described herein,wherein the immunotherapeutic agent is a tumor antigen vaccine oranti-tumor dendritic cell vaccine.

Another aspect of the invention relates to the use of a compounddescribed herein in the manufacture of a medicament for the treatment ofa disorder for which inhibition of proteasome activity providestherapeutic benefit.

Another aspect of the invention relates to a packaged pharmaceutical,comprising a prodrug described herein formulated in a pharmaceuticallyacceptable excipient, in association with instructions (written and/orpictorial) describing the recommended dosage and/or administration ofthe formulation to a patient.

In certain embodiments, the compounds and compositions of the inventionmay also be combined with chemotherapy. The efficacy of chemotherapy—amainstay of the standard of care in carcinoma—is limited bychemoresistance due to the activation of NF-κB by chemotherapeuticagents, resulting in inhibition of the apoptotic response of tumorcells. Tumor cells also resist chemotherapy by overexpression of Bcl-2and P-glycoprotein. Proteasome inhibitors (PIs) counter these effects byrepressing activation of NF-κB, inducing cleavage of Bcl-2 intoproapoptotic fragments, and preventing maturation of P-glycoprotein intothe active form that removes chemotherapeutic agents from the cancercell. Therefore, PIs could act as adjuvants to chemotherapy. Compared tobortezomib in this role, the compounds and compositions disclosed hereinmay reduce compounded toxicities: e.g., increased grade 3/4 hematologictoxicity associated with bortezomib plus gemcitabine, docetaxel orcarboplatin. Chemotherapeutic agents are usually administered at highdoses in cycles interspersed with breaks. More continuous administrationof chemotherapeutic agents (metronomic chemotherapy) has recently beeninitiated in order to lengthen exposure of cancer cells to drug andinhibit angiogenesis. Due to reduced toxicity, the compounds andcompositions disclosed herein would be ideally suited for longer periodsof administration in combination with metronomic chemotherapy.

Definitions

The term “amino acid” is intended to encompass all compounds, whethernatural or synthetic, which include both an amino functionality and anacid functionality, including amino acid analogues and derivatives. Incertain embodiments, the amino acids contemplated in the presentinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids, which contain amino and carboxyl groups. Naturally occurringamino acids are identified throughout by the conventional three-letterand/or one-letter abbreviations, corresponding to the trivial name ofthe amino acid, in accordance with the following list. All amino acidsdescribed herein are contemplated as both (D)- and (L)-isomers unlessotherwise designated. The abbreviations are accepted in the peptide artand are recommended by the IUPAC-IUB commission in biochemicalnomenclature.

By the term “amino acid residue” is meant an amino acid. In general theabbreviations used herein for designating the naturally occurring aminoacids are based on recommendations of the IUPAC-IUB Commission onBiochemical Nomenclature. See Biochemistry (1972) 11:1726-1732). Forinstance Met, Ile, Leu, Ala and Gly represent “residues” of methionine,isoleucine, leucine, alanine and glycine, respectively. By the residueis meant a radical derived from the corresponding α-amino acid byeliminating the OH portion of the carboxyl group and the H portion ofthe α-amino group.

The term “amino acid side chain” is that part of an amino acid residueexclusive of the backbone, as defined by K. D. Kopple, “Peptides andAmino Acids”, W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2and 33; examples of such side chains of the common amino acids are—CH₂CH₂SCH₃ (the side chain of methionine), —CH₂(CH₃)—CH₂CH₃ (the sidechain of isoleucine), —CH₂CH(CH₃)₂ (the side chain of leucine) or H—(the side chain of glycine). These sidechains are pendant from thebackbone Cα carbon.

The term “amino acid analog” refers to a compound structurally similarto a naturally occurring amino acid wherein the C-terminal carboxygroup, the N-terminal amino group or side-chain functional group hasbeen chemically modified. For example, aspartic acid-(beta-methyl ester)is an amino acid analog of aspartic acid; N-ethylglycine is an aminoacid analog of glycine; or alanine carboxamide is an amino acid analogof alanine

The phrase “protecting group” as used herein means substituents whichprotect the reactive functional group from undesirable chemicalreactions. Examples of such protecting groups include esters ofcarboxylic acids and boronic acids, ethers of alcohols, and acetals andketals of aldehydes and ketones. For instance, the phrase “N-terminalprotecting group” or “amino-protecting group” as used herein refers tovarious amino-protecting groups which can be employed to protect theN-terminus of an amino acid or peptide against undesirable reactionsduring synthetic procedures. Examples of suitable groups include acylprotecting groups such as, to illustrate, formyl, dansyl, acetyl,benzoyl, trifluoroacetyl, succinyl, and methoxysuccinyl; aromaticurethane protecting groups as, for example, benzyloxycarbonyl (Cbz); andaliphatic urethane protecting groups such as t-butoxycarbonyl (Boc) or9-Fluorenylmethoxycarbonyl (Fmoc).

The term “amino-terminal protecting group” as used herein, refers toterminal amino protecting groups that are typically employed in organicsynthesis, especially peptide synthesis. Any of the known categories ofprotecting groups can be employed, including acyl protecting groups,such as acetyl, and benzoyl; aromatic urethane protecting groups, suchas benzyloxycarbonyl; and aliphatic urethane protecting groups, such astert-butoxycarbonyl. See, for example, Gross and Mienhoffer, Eds., ThePeptides, Academic Press: New York, 1981; Vol. 3, 3-88; and Green, T.W.; Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd ed,Wiley: New York, 1991. Preferred protecting groups include aryl-,aralkyl-, heteroaryl- and heteroarylalkyl-carbonyl and sulfonylmoieties.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable mammalian cell

The term “prodrug” as used herein encompasses compounds that, underphysiological conditions, are converted into therapeutically activeagents. A common method for making a prodrug is to include selectedmoieties that are hydrolyzed under physiological conditions to revealthe desired molecule. In other embodiments, the prodrug is converted byan enzymatic activity of the host animal.

The phrase “pharmaceutically acceptable excipient” or “pharmaceuticallyacceptable carrier” as used herein means a pharmaceutically acceptablematerial, composition or vehicle, such as a liquid or solid filler,diluent, excipient, solvent or encapsulating material, involved incarrying or transporting the subject chemical from one organ or portionof the body, to another organ or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation, not injurious to the patient, andsubstantially non-pyrogenic. Some examples of materials which can serveas pharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations. In certain embodiments, pharmaceutical compositions of thepresent invention are non-pyrogenic, i.e., do not induce significanttemperature elevations when administered to a patient.

The term “pharmaceutically acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, naphthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts, and the like.See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19.

In other cases, the compounds useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts, and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like(see, for example, Berge et al., supra).

A “therapeutically effective amount” of a compound with respect to usein treatment, refers to an amount of the compound in a preparationwhich, when administered as part of a desired dosage regimen (to amammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “self-eliminating linker” or “self-immolative linker” refers toa temporary extender, spacer, or placeholder unit attaching two or moremolecules together by chemical bonds that are cleaved under definedconditions to release the two molecules. Examples of self-eliminatinglinkers include, but are not limited to, p-aminobenzyloxycarbonyl (PABC)and 2,4-bis(hydroxymethyl)aniline. The self-eliminating orself-immolative linker may be linear or branched, and may link two ormore of the same molecules together, or may link two or more differentmolecules together. The self-eliminating or self-immolative linker maydegrade, decompose, or fragment under, for example, physiologicalconditions, acidic conditions, basic conditions, or in the presence ofspecific chemical agents.

As noted above, certain compounds of the present invention may exist inparticular geometric or stereoisomeric forms. The present inventioncontemplates all such compounds, including cis- and trans-isomers, R-and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomer. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomer.

An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyldefined below. A straight aliphatic chain is limited to unbranchedcarbon chain moieties. As used herein, the term “aliphatic group” refersto a straight chain, branched-chain, or cyclic aliphatic hydrocarbongroup and includes saturated and unsaturated aliphatic groups, such asan alkyl group, an alkenyl group, or an alkynyl group.

“Alkyl” refers to a fully saturated cyclic or acyclic, branched orunbranched carbon chain moiety having the number of carbon atomsspecified, or up to 30 carbon atoms if no specification is made. Forexample, alkyl of 1 to 8 carbon atoms refers to moieties such as methyl,ethyl, propyl, butyl, pentyl, hexyl, heptyl, and octyl, and thosemoieties which are positional isomers of these moieties. Alkyl of 10 to30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl,heneicosyl, docosyl, tricosyl and tetracosyl. In certain embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer.

“Cycloalkyl” means mono- or bicyclic or bridged saturated carbocyclicrings, each having from 3 to 12 carbon atoms. Likewise, preferredcycloalkyls have from 5-12 carbon atoms in their ring structure, andmore preferably have 6-10 carbons in the ring structure.

Unless the number of carbons is otherwise specified, “lower alkyl,” asused herein, means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths. Throughout the application,preferred alkyl groups are lower alkyls. In certain embodiments, asubstituent designated herein as alkyl is a lower alkyl.

“Alkenyl” refers to any cyclic or acyclic, branched or unbranchedunsaturated carbon chain moiety having the number of carbon atomsspecified, or up to 26 carbon atoms if no limitation on the number ofcarbon atoms is specified; and having one or more double bonds in themoiety. Alkenyl of 6 to 26 carbon atoms is exemplified by hexenyl,heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodenyl, tridecenyl,tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl,nonadecenyl, eicosenyl, heneicosoenyl, docosenyl, tricosenyl, andtetracosenyl, in their various isomeric forms, where the unsaturatedbond(s) can be located anywherein the moiety and can have either the (Z)or the (E) configuration about the double bond(s).

“Alkynyl” refers to hydrocarbyl moieties of the scope of alkenyl, buthaving one or more triple bonds in the moiety.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur moiety attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —(S)-alkyl, —(S)-alkenyl,—(S)-alkynyl, and —(S)—(CH₂)_(m)—R¹, wherein m and R¹ are defined below.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen moiety attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propoxy,tert-butoxy, and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R¹,where m and R₁ are described below.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the formulae:

wherein R³, R⁵ and R⁶ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R¹, or R³ and R⁵ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R¹ represents an alkenyl, aryl, cycloalkyl,a cycloalkenyl, a heterocyclyl, or a polycyclyl; and m is zero or aninteger in the range of 1 to 8. In certain embodiments, only one of R³or R⁵ can be a carbonyl, e.g., R³, R⁵, and the nitrogen together do notform an imide. In even more certain embodiments, R³ and R⁵ (andoptionally R⁶) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R¹. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted orunsubstituted alkyl attached thereto, i.e., at least one of R₃ and R₅ isan alkyl group. In certain embodiments, an amino group or an alkylamineis basic, meaning it has a conjugate acid with a pK_(a)≧7.00, i.e., theprotonated forms of these functional groups have pK_(a)s relative towater above about 7.00.

The term “aryl” as used herein includes 3- to 12-membered substituted orunsubstituted single-ring aromatic groups in which each atom of the ringis carbon (i.e., carbocyclic aryl) or where one or more atoms areheteroatoms (i.e., heteroaryl). Preferably, aryl groups include 5- to12-membered rings, more preferably 6- to 10-membered rings The term“aryl” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining ringswherein at least one of the rings is aromatic, e.g., the other cyclicrings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls. Carboycyclic aryl groups includebenzene, naphthalene, phenanthrene, phenol, aniline, and the like.Heteroaryl groups include substituted or unsubstituted aromatic 3- to12-membered ring structures, more preferably 5- to 12-membered rings,more preferably 6- to 10-membered rings, whose ring structures includeone to four heteroatoms. Heteroaryl groups include, for example,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to12-membered ring structures, more preferably 5- to 12-membered rings,more preferably 6- to 10-membered rings, whose ring structures includeone to four heteroatoms. Heterocycles can also be polycycles.Heterocyclyl groups include, for example, thiophene, thianthrene, furan,pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole,imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring can be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, and the like.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the formula:

wherein X is a bond or represents an oxygen or a sulfur, and R⁷represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R¹ or apharmaceutically acceptable salt, R⁸ represents a hydrogen, an alkyl, analkenyl or —(CH₂)_(m)—R¹, where m and R¹ are as defined above. Where Xis an oxygen and R⁷ or R⁸ is not hydrogen, the formula represents an“ester.” Where X is an oxygen, and R⁷ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R⁷ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen, and R⁸ is a hydrogen, the formula represents a “formate.” Ingeneral, where the oxygen atom of the above formula is replaced by asulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R⁷ or R⁸ is not hydrogen, the formula represents a“thioester” group. Where X is a sulfur and R⁷ is a hydrogen, the formularepresents a “thiocarboxylic acid” group. Where X is a sulfur and R⁸ isa hydrogen, the formula represents a “thioformate” group. On the otherhand, where X is a bond, and R⁷ is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R⁷ is a hydrogen,the above formula represents an “aldehyde” group.

The term “thioxamide,” as used herein, refers to a moiety that can berepresented by the formula:

in which R^(t) is selected from the group consisting of the groupconsisting of hydrogen, alkyl, cycloalkyl, aralkyl, or aryl, preferablyhydrogen or alkyl. Moreover, “thioxamide-derived” compounds or“thioxamide analogues” refer to compounds in which one or more amidegroups have been replaced by one or more corresponding thioxamidegroups. Thioxamides are also referred to in the art as “thioamides.”

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds. It will be understood that “substitution” or “substitutedwith” includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br, or —I; the term “sulfhydryl” means —SH; theterm “hydroxyl” means —OH; the term “sulfonyl” means —SO₂—; the term“azido” means —N₃; the term “cyano” means —CN; the term “isocyanato”means —NCO; the term “thiocyanato” means —SCN; the term “isothiocyanato”means —NCS; and the term “cyanato” means —OCN.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the formula:

in which R³ and R⁵ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the formula:

in which R⁷ is as defined above.

The term “sulfonamide” is art recognized and includes a moiety that canbe represented by the formula:

in which R³ and R⁸ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the formula:

in which R⁷ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the formula:

in which R¹² is selected from the group consisting of the groupconsisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,heterocyclyl, aralkyl, or aryl.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th ed., 1986-87, inside cover.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 ARI-3996 and Proteasome Inhibitors of the Invention

ARI-3996 and its PI warhead ARI-2727D were synthesized as shown in FIG.1, using synthetic and analytical methods previously described forobtaining peptide boronic acids. Bortezomib was purchased from SelleckChemicals or ChemieTek. Each batch of ARI-3996 was validated forselective cleavage by FAP versus PREP as described herein.

The following reagents were used: (a) HATU/DMF/DIPEA, 95% yield; (b) 4 MHCl in dioxane, 100% yield; (c) HATU/DMF/DIPEA, 90% yield; (d) 4M HCl indioxane, 100% yield; (e) tBu-Suc-Ser(tBu)-OH, HATU/DMF/DIPEA, 90% yield;(f) Pd(OH)₂—C/H₂/methanol, 90% yield; (g) 2727D, HATU/DMF/DIPEA, 85%yield; (h) TFA/DCM, 90% yield; (i) PhB(OH)₂, pentane-water-acetonitrile,70% yield. The chemical synthesis of peptides, particularly shortpeptides such as di- and tripeptides such as those described herein, iswell-known in the art and sufficiently predictable due to its modularnature. Therefore the synthetic methods of Table 1 or standardsolid-phase synthetic methods as applied to peptides would be successfulin delivering any compound of formula I.

ARI-3996 is a pro-drug version of a bortezomib-like cytotoxic agentdesigned to more selectively target the proteasome in solid tumors (FIG.15). ARI-3996 was designed to reduce the mechanism-based DLTs associatedwith proteasome inhibition by remaining inactive until it is cleaved byfibroblast activation protein (FAP) on the surface of reactive stromalfibroblasts of epithelial tumors. Because FAP is produced in epithelialtumors but not usually in healthy tissues (FAP is expressed in thestroma of many common epithelial tumors—lung, colon, breast andpancreatic cancer), ARI-3996 should not be activated in nervous tissueor in bone marrow where platelets are generated. ARI-3996 is relativelynon-toxic to all cells and cannot kill tumor cells until it is activatedby FAP. Therefore, ARI-3996 should kill FAP-producing tumors with lesssevere PN and thrombocytopenia than that associated with bortezomib.

Fibroblast activation protein (FAP) is a post-prolyl cleaving serineprotease belonging to the (dipeptidyl peptidase) DPP-IV-like subfamilyin which FAP and prolyl endopeptidase (PREP) are the only mammalianproteases that can cleave on the C-terminal side of internal proline.Unlike FAP, PREP is constitutively and ubiquitously expressed. We havesolved the specificity problem of FAP versus PREP cleavage required tomake a pro-drug selectively activated by FAP. FAP's P₄-P₁ cleavagespecificity requires proline at P₁, glycine or D-amino acids at P₂,prefers small uncharged amino acids at P₃, and tolerates most aminoacids at P₄. We have discovered that D-alanine at P₂ allows cleavage byFAP as expected but prevents cleavage by PREP. Thus, linkage of thetripeptide Suc-Ser-D-Ala-Pro by a C-terminal peptide bond to thebortezomib-like aminoboronate dipeptide Ala(1-NaI)-boroLeu (ARI-2727D)produces the pro-drug ARI-3996 in which the proteasome inhibitoryactivity is unleashed selectively by FAP cleavage of the Pro-Ala(1-NaI)bond. In vitro, ARI-3996 is cleaved by FAP, but to a far lesser extentby PREP, to yield the cytotoxic “warhead” ARI-2727D as demonstrated bymass spectrometry (FIG. 2) and by assay of tumor-cell killing in vitroby fluorescent cell-titer blue (Promega) (Table 1).

TABLE 1 FAP specifically activates ARI-3996 to kill tumor cells in vitroCytotoxic EC₅₀ ^(a) (μM) of ARI-3996 incubated with^(b): Cell line NoneFAP PREP RPMI 8226 5.8 0.18 3.1 KG-1 22.0 0.30 13.0 RPMI 1788 6.2 0.133.6 BxPC-3 34.0 0.80 21.0 ^(a)Drug concentration that kills 50% ofcells. ^(b)24 hours at 37° C., except BxPC-3, 48 hours

Example 2 Use of ARI-3144 as a Diagnostic/Patient Stratification Tool

The structure of ARI-3144 is shown above and includes an arrowindicating the chemical bond that is cleaved by FAP. FIG. 11 shows acartoon of the concept underlying the diagnostic use of this compound.The FAP recognition site is chemically attached to the fluorogeniccoumarin (AMC) moiety. After binding of ARI-3144 to FAP, chemical bondcleavage takes place (FIG. 12) to release the FAP recognition site fromthe AMC. Once released, AMC is now fluorescent and its concentration canbe quantified by measuring its spectroscopic properties.

The D-alanine at P₂ of the fluorogenic substrate,N-(Quinoline-4-carbonyl)-D-Ala-Pro-AMC [AMC=7-amino-4-methylcoumarin](ARI-3144), confers selectivity for FAP so that it is cleaved to releasefluorescent AMC by recombinant FAP but not by other DPP-IV-likeproteases (FIG. 3). As shown in FIG. 13, ARI-3144 is an excellentsubstrate for FAP, and no cleavage by PREP, DPPIV, DPP8, DPP9, or DPPIIwas detected (FIG. 14). FAP is reportedly not expressed constitutivelyin healthy tissue with the exception of endometrium, although FAPproteolytic activity is detectable in plasma. Ovarian and prostatetumors excepted, induction of FAP expression in stromal fibroblasts ofcommon epithelial tumors (lung, colon, breast and pancreas) has beendemonstrated by immunohistochemistry and mRNA analysis. The ability tomeasure FAP proteolytic activity in tumors is required to evaluate theusefulness of ARI-3996 as a chemotherapeutic agent. Hitherto, FAPactivity has not been quantified in human or mouse tumors. We have usedthe ARI-3144 assay ex vivo to demonstrate increased FAP proteolyticactivity in human pancreatic carcinoma relative to healthy tissues andplasma. We will use the assay to select a mouse tumor model forinvestigation of ARI-3996's activity in which the increase intumor-associated FAP activity relative to plasma is equivalent to thatin human epithelial cancer.

Besides coumarin-based chromophores, other widely-used chromophoreswould work just as well as long as an amide linkage could be used forattachment (i.e., the chromophore has a primary amino group availablefor attachment to the proline of the FAP recognition site. Suchcommonly-used chromophores are, for example:

Example 3 Mouse Model of Epithelial Cancer in which the FAP ProteolyticActivity of Tumor Tissue is Equivalent to that in Human Cancer Patients

The FAP-specific fluorogenic substrate ARI-3144 was used to measure FAPproteolytic activity in tissue homogenates and plasma in a standardcontinuous fluorometric assay as previously described.

In tissue specimens from 14 pancreatic cancer patients at the Fox ChaseCancer Center, we determined mean (±SE) FAP activity in cancerous tissueof 903.7±161.4 expressed as change in fluorescent units (AFU)/min/mgprotein. FAP activity varied between patients with 4 high-expressersthat were in the range of 1,200 to 3,000. In contrast, HPAF-IIpancreatic adenocarcinoma xenografts in scid mice exhibited meanactivity of only 200±12.5. In mice, we have found that circulatinglevels of plasma FAP activity are approximately 6-fold higher than inhumans regardless of whether either species bears a tumor. Thus, thetumor:plasma ratio of FAP activity is at least 100:1 in humans; but only3:1 in the HPAFII xenografted mice (FIGS. 4 and 16).

The TI of a FAP-activated pro-drug such as ARI-3996 is expected todepend on the difference between the systemic level of FAP proteolyticactivity and the level in tumor tissue. ARI-3996 has exhibitedsignificant antitumor activity in HPAF-II mice (see below). However, theHPAF-II model does not accurately reflect the tumor:systemic ratio ofFAP activity in human pancreatic carcinoma patients (FIGS. 16 and 17).In order to test the feasibility of ARI-3996 as a safer and moreeffective PI than bortezomib in solid cancer, a carcinoma model in whichtumor FAP activity is ˜35-fold higher than in HPAF-II xenografts isneeded. Because FAP is induced in reactive stromal fibroblasts duringtumorigenesis, the level of tumor FAP activity should be higher in mousemodels that recapitulate the pattern of stromal development in humancancer than in xenografts of cell lines. Two different models appearpromising. In the Cre-recombinase inducible lung adenocarcinoma model inLox-Stop-Lox (LSL)-K-ras^(G12D) mice, endogenous tumor developmentinduces a FAP⁺ stroma that closely resembles that in human carcinomahistologically. An alternative model is provided by patient tumorsdirectly transplanted into immunodeficient mice. The transplanted humantumors are reported to maintain the stromal organization and vasculatureof the original tumor. FAP activity will be assayed in samples ofxenograft transplants of human epithelial carcinomas with well-developedstroma provided by Oncotest http://www.oncotest.de/for-pharma/index.php.

As shown in FIG. 5, the FAP transfected HEK293 xenograft model can beused to model human tumors overexpressing FAP to a similar degree as intumors found in human cancer patients. A FAP-transfected variant of theHEK293 cell line forms tumors of FAP′ epithelial cells in scid mice(69). We have demonstrated FAP activity levels of 6,000 to 12,500ΔFU/min/mg in FAP-HEK293 tumors in vivo (FIG. 5). The FAP-HEK293 modelis, therefore, suitable for investigation of ARI-3996's TI. However,unlike the K-ras^(G12D)-driven lung tumor and direct patient transplantmodels, the HEK293 model does not mimic the stromal expression of FAP inhuman carcinoma.

Furthermore, as shown in FIG. 18, FAP transfected HEK tumor xenograftshave FAP activity matching human pancreatic cancer tumors. A battery ofhuman pancreatic tumor samples had FAP activity levels ranging fromnegligible to over 250 (tumor FAP/plasma FAP). The right hand side ofFIG. 18 shows that HEK mouse xenograft samples display ratios of tumorFAP/plasma FAP from 150-270.

Example 4 Validation of the HEK Tumor Xenograft Mouse Model

Having determined the levels of FAP expression in the HEK xenograftmodel, the anticancer activity of ARI-3996 was next evaluated in a40-day study. FIG. 20 shows the results. Impressively, while Velcade®hardly slowed the growth of the tumors, both ARI-2727D and ARI-3996exhibited potent tumor inhibition. ARI-2727D is expected to be lesspotent than ARI-3996 because it lacks the FAP recognition site, oraddress moiety. It also suffers conformation-dependent inactivation overtime. Nonetheless, ARI-2727 showed a markedly greater inhibitory effectthan Velcade®.

At a dose of 25 mg/kg, ARI-3996 showed nearly complete inhibition oftumor growth. Even in an HEK-mock model (FIG. 20 bottom) at a dose of 50mg/kg ARI-3996 was well tolerated and showed an inhibitory effect overthe control.

As a further test of its efficacy, ARI-3996 was administered toimmunocompetent WT BALB/c mice. As FIG. 19 shows, tumor regression wasobserved over the course of a 30-day experiment.

Example 5 Maximum Tolerated Doses (MTD) and Minimum Effective Doses(MED) of ARI-3996, ARI-2727D, and Bortezomib in the FAP⁺ Cancer Model

ARI-3996 administered (i.p.) to mice xenografted with the HPAF-II cellline significantly reduced tumor growth at its MTD of 100 mg/kg (FIG.6). The antitumor effect of ARI-3996 was confirmed both as a singleagent and in combination with gemcitabine (FIGS. 9 & 10). In particular,highly significant antitumor effect was observed when ARI-3996 wasadministered s.c. instead of i.p. (FIG. 9). In contrast, HPAF-II tumorswere refractory to bortezomib at its MTD of 1 mg/kg (FIG. 6). ARI-3996,therefore, appears to be 100-fold safer than bortezomib based on MTD andto outperform bortezomib in a model of epithelial cancer. However, theantitumor effect of ARI-3996 was likely limited by the relatively lowlevel of FAP activity, which is required to activate the prodrug, inHPAF-II tumors. As described above, the FAP tumor:plasma ration is≧100:1 for human pancreatic cancer versus 3:1 in HPAF-II xenograftedmice (FIG. 4). Therefore, in order to better judge ARI-3996's potentialfor producing antitumor effects in carcinoma patients at tolerated doselevels, MTDs and MEDs of ARI-3996, bortezomib and ARI-2727D will becompared in the mouse model selected for tumor-associated FAP activityequivalent to that in human cancer.

MTDs will be determined by administering (i.p.) escalating doses ofcompounds twice weekly (days 1 and 4) to groups of normal andtumor-bearing mice (n=2 female+2 male). Comparison of toxicity intumor-bearing versus non-tumor-bearing mice will determine whetheractivation of ARI-3996 by tumor FAP contributes to systemic toxicity.Health of mice will be monitored daily, and mice will be weighed twiceweekly. At sublethal dose levels, the highest dose that causes no illhealth and no greater than 10% weight loss will be defined as the MTD.MEDs will be determined from dose responses of antitumor effects intumor-bearing mice (n=5-7 per treatment group) administered compoundstwice weekly. The MED will be defined as the smallest dose that producesa significant reduction in tumor growth as determined by unpaired,two-tailed Student's t test for comparison of tumor sizes between testand control mice. Experimental details will depend on the model chosenin Experiment 1. Study design will be similar to that previouslydescribed for the demonstration of the antitumor effect of theFAP-targeting antitumor agent, Glu-boroPro. TIs for ARI-3996, bortezomiband the ‘warhead’, ARI-2727D, will be calculated by the formula:TI=MTD÷MED. If the availability of LSL-K-ras^(G12D) or Oncotest mice islimiting, toxicity and MTD can be investigated in FAP-HEK293 xenograftedscid mice (FIG. 5).

Example 6 Characterization of the Mechanism of the Antitumor Effect ofARI-3996 by Investigating Inhibition of the 20S Proteasome, Induction ofApoptosis, and Reduction of Angiogenesis in FAP Tumors

One hour after final drug administration at termination of Example 5,peripheral blood, tumor, spleen and liver will be collected. Tissuelysates will be prepared from snap-frozen samples for assay ofproteasome inhibition. Histological tissue specimens will be fixed informalin and embedded in paraffin under conditions suited toimmunostaining and apoptosis assay and sectioned.

Chymotryptic subunit activity of the 20S proteasome will be determinedusing the fluorogenic substrate succ-Leu-Leu-Val-Tyr-AMC (Enzo LifeScience). Bortezomib and ARI-2727D will distribute to all tissues andare expected to inhibit proteasome activity in all tissues, uniformly,in a dose-dependent manner. The assay will test whether ARI-3996 targetstumor proteasome activity more selectively using a paired, two-tailedStudent's t test for comparisons between paired samples of tumor andnon-tumor tissue (e.g., spleen) in each animal. Histological sections oftumors that responded optimally to drug treatments will be compared tocontrols for microvessel density (MVD) by immunostaining with mouseCD34-specific antibody (BD-Pharmingen). Apoptosis will be quantified byterminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL)using the ApopTag peroxidase in situ apoptosis detection kit(Millipore). Events will be counted microscopically in a blinded manner,and significance of differences between vehicle- and drug-treated tumorswill be determined by unpaired, two-tailed Student's t test for at least5 mice per treatment group. Tissues will also be stained with H&E forinvestigation of systemic toxicity.

We have found that FAP proteolytic activity of blood plasma appears tobe approximately 6-fold higher in mice (˜ΔFU/min) than in humans(˜ΔFU/min) regardless of tumor status. Mouse models may over-reportsystemic toxicity of ARI-3996 compared with that possible in cancerpatients. This was further verified by treatment of FAP knockout micewith ARI-3996. In knockout mice (FIG. 21), no activation of ARI-3996 torelease ARI-2727 took place, whereas in FAP+ mice a concentration of thereleased warhead ARI-2727 of 150 ng/mL was reached.

Greater pro-drug activation in the peripheral blood of mice could resultin greater systemic exposure to the ARI-2727D “warhead” than in humans.If mouse toxicity prevents achievement of the targeted 10-fold greaterTI for ARI-3996 versus bortezomib, we will investigate toxicity in micethat are genetically deficient in FAP (Fap^(LacZ/LacZ)) (70). We havedemonstrated that FAP-deficient mice have no significant proteolyticactivity detectable with the FAP-specific substrate ARI-3144 (FIG. 1).Therefore, comparison of the MTD of ARI-3996 in FAP-sufficient versusFAP-deficient mice will determine how plasma FAP activity affectstoxicity. If we find that ARI-3996 has highly significant preclinicalantitumor activity, but the TI is compromised due to the basal level ofplasma FAP activity in mice, we would consider the Test of Feasibilityto be met.

Example 7 Evidence for Tumor Delivery of ARI-2727D by the FAP ActivatedProdrug ARI-3996

Dose-limiting toxicity (DLT) prevents administration of high-enoughdoses of bortezomib to produce tumor responses in solid cancer.Preclinical results in mice xenografted s.c. with the human PC-3prostate tumor suggest that DLT is due to the low exposure of solidtumors to bortezomib relative to the exposure of non-cancerous tissue(FIG. 7). ARI-3996 is a pro-drug designed to release a bortezomib-likePI, ARI-2727D, at the tumor site upon cleavage by proteolytic activityof fibroblast activation protein (FAP). Because FAP is predominantlyexpressed in stroma of human epithelial cancer, ARI-3996 should increasetumor exposure to the PI and reduce exposure in healthy tissues relativeto bortezomib.

In SCID mice xenografted with the human HPAF-II pancreaticadenocarcinoma, we have compared the tissue distribution of ARI-3996 andARI-2727D in liver, peripheral blood cells (PBC) and tumor following asingle s.c. injection of ARI-3996 at a dose of 50 mg/kg. At 1, 3 and 6hours after administration, as for bortezomib (FIG. 7), liver exposureto intact ARI-3996 is greater than tumor exposure (FIG. 8 A, C, E), andexposure of PBC and tumor to pro-drug is similar at 3 hours. However, atall time points, tumor exposure to the active “warhead”, ARI-3996exceeded either liver or PBC exposures (FIG. 8 B, D, F).

Further evaluation (FIGS. 22 and 23) of the tissue distribution ofVelcade® vs. ARI-3996 in mice showed that Velcade® reached much higherconcentrations in the heart, lung, kidney, liver, spleen, and lung thanin the tumor, suggesting that the drug's ineffectiveness against solidtumors results from its low concentration in the tumor. Velcade®'s hightoxicity would also result from the accumulation of the drug in theorgans at the expense of accumulation in the tumor. In contrast,ARI-3996 accumulates primarily in the liver initially; FAPactivation/cleavage to form ARI-2727D results in ARI-2727D forming muchhigher relative concentrations in the tumor with lesser amounts in thelungs and plasma. Thus ARI-2727D is being selectively delivered to solidtumors via FAP activation of ARI-3996.

Finally, FAP activation was verified as the mode by which ARI-2727Dtumor accumulation was taking place. In FIG. 24, ARI-2727Dconcentrations at 1 hour post-injection were compared with ARI-2727Dconcentrations 1 hour after injection of the prodrug form (ARI-3996).Direct injection of ARI-2727D resulted in the highest concentration ofthe drug accumulating in the kidneys and lung; when ARI-3996 wasinjected the highest concentration of ARI-2727D was found in the tumor,followed by lungs and plasma.

Remarkably, the results suggest that ARI-3996 increases tumor exposureto the active PI while sparing non-tumor tissue. Interestingly, in theHPAF-II tumor model, we have found that bortezomib lacks significantantitumor activity at the maximum tolerated dose of 1 mg/kg in mice(FIG. 6), whereas ARI-3996 is well tolerated and produces significantreductions in tumor size at 50 mg/kg (FIGS. 9 and 10). The HPAF-II tumorresponse to ARI-3996 strengthens our hypothesis that solid tumors canrespond to proteasome inhibition. The tumor:plasma ratio of FAP activityis only 3:1 in HPAF-II mice, whereas in pancreatic cancer patients theratio is 100:1 or greater. Therefore, we anticipate significantlygreater activation of ARI-3996 and, consequently, further improvementsin tumor responses in the mouse model with a higher tumor:plasma FAPwill be identified in further studies.

Example 8 Cytotoxicity of Velcade® Versus ARI-2727D and ARI-3996 inMultiple Myeloma, Normal Cells, and Solid Tumors

Although Velcade® has robust clinical activity in MM patients, drugresistance develops in all patients who initially respond to treatment.Stromal fibroblasts in epithelial tumors promote tumor progression andmetastasis through the remodeling of the extracellular matrix and as asource of paracrine growth factors such as fibroblast growth factor,epidermal growth factor and transforming growth factor-β. By targetingproteasome inhibition to the tumor microenvironment, ARI-3996 may killstromal fibroblasts as well as malignant epithelial cells. This wouldprovide the opportunity to attack the tumor by killing a cell type thatis less likely than the tumor cell itself to develop drug-resistance.

FIGS. 25 and 26 demonstrate further biological evaluation of ARI-2727Dand ARI-3996 vs. Velcade® for cytotoxicity toward Multiple Myeloma (MM),normal cells, and various solid tumors. FIG. 25 shows that both Velcade®and ARI-2727D have extremely high potency against various MM cell linesand slightly lower toxicity against normal cells. Their toxicity is muchlower against solid tumors. In FIG. 26 ARI-3996 is compared withARI-2727D and Velcade®. Its cytotoxicity is much lower across the board,particularly in solid tumors.

These results underscore the importance of selective delivery in solvingthe ongoing challenges in conventional cancer chemotherapy. Withoutselective delivery of cytotoxic agents to cancer cells, they oftendisplay equal toxicity to normal and cancerous cells alike.

Example 9 FAP Activity of Human Cancers

One important aspect of determining which cancers will benefit fromtreatment with the compounds of the invention. As mentioned above, FAPhas very low expression in normal human tissues. A large number oftissue samples from tumors were collected and their FAP activity—notexpression levels—measured. As FIG. 27 shows, virtually all the samplesshow a much higher level of FAP activity in the tumor vs. the serum.Thus, most solid tumors susceptible to proteasome inhibitors areexpected to respond to treatment with FAP-activated prodrugs of theinvention. As shown in FIG. 4, human tumors have, on average, 100:1 theFAP activity levels of normal human tissue.

Example 10 Anticancer Effects of Proteasome Inhibitors in U266Tumor-Bearing Mice

ARI-3996 consistently outperformed Velcade® in a mouse MM model. Micebearing U266 tumor xenografts (2 female, 2 male) were treated witheither the vehicle, ARI-3996, or Velcade® twice a week (day 1 and day 4)for 2 weeks. As shown in FIG. 28, ARI-3996 was dosed at 50 mg/kg (½ theMTD) and Velcade® at 0.5 mg/kg (also ½ the MTD). The inhibition of thetumor was evaluated using ELISA (μg/mL) and bioluminescence. ARI-3996showed a marked advantage over Velcade®. While 1 death took place in theVelcade® group, all mice in the ARI-3996 group survived with improvedoutcomes vs. the Velcade® group.

Example 11 Conjugation of FAP Recognition Site to Known ProteasomeInhibitors

Since the above Examples demonstrate that the FAP recognition site (theshort peptide chain conferring FAP specificity) when attached toARI-2727D confers selective delivery of the warhead to tumors and thesurrounding stromal cells, it is reasonable to conclude that the sameFAP recognition sequence could be attached to other proteasomeinhibitors to yield the same effect. Many short peptide and peptideanalogue sequences are known to inhibit the proteasome. Attachment ofthese sequences to the FAP recognition site by the N-terminal amide ofthe inhibitor/warhead will form prodrug of similar potency, specificity,and (low) toxicity as ARI-3996.

Many of the most potent proteasome inhibitors contain 2-4 peptides orpeptide analogues with an electrophilic moiety replacing or appended tothe carboxyl terminus. This electrophilic moiety is a reactive speciesthat covalently modifies a nucleophilic residue of the proteasome,destroying its catalytic activity. Such a method of inactivating anenzyme is commonly referred to as “suicide inhibition” in theliterature. Examples of successfully validated electrophilic moietiesinclude boronates, epoxyketones, aldehydes, cyanates, vinyl sulfones,α,β-unsaturated carbonyls, and ketoaldehydes.

The structures of a number of clinically relevant or otherwise validatedproteasome inhibitors are shown in FIG. 29. Careful consideration of thechemical structures reveals certain similarities. Most are di-, tri-, ortetrapeptides with an electrophilic moiety attached to the carboxylterminus. At the amino terminus is generally an acyl or aracyl group(bortezomib, CEP-18770, MLN2238, MLN9708, MG-132, PSI, ¹²⁵I-NIP-L₃VS,carfilzomib, oprozomib, epoximicin, PR-957, NC-005, NC-005-VS, YU-101,LU-005, YU-102, NC-001, NC-022, CPSI, and IPSI-001). These acyl oraracyl groups are present to increase the resistance of the proteasomeinhibitor against nonspecific proteases that might otherwise degradeshort peptides.

If these N-terminal acyl or aracyl groups attached to the variousproteasome inhibitors shown in FIG. 29 are removed and replaced with theFAP recognition site described herein, the result would be novelFAP-activated proteasome inhibitors whose specificity and toxicity wouldbe greatly improved over their parent molecules.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

We claim:
 1. A fibroblast activation protein (FAP)-activated proteasomeinhibitor represented by formula II:

wherein R₁—(C═O)— represents an acyl N-terminal blocking group; R₂represents H, lower alkyl, or a mono- or di-hydroxy-substituted loweralkyl; R₃ represents H, halogen, or lower alkyl; R₄ is absent orrepresents lower alkyl, —OH, —NH₂ or halogen; and

is selected from the group consisting of:


2. A FAP-activated proteasome inhibitor represented by formula III:

wherein R₁—(C═O)— represents an acyl N-terminal blocking group; R₂represents H, lower alkyl, or a mono- or di-hydroxy-substituted loweralkyl; R₃ represents H, halogen, or lower alkyl; R₄ is absent orrepresents lower alkyl, —OH, —NH₂ or halogen; R₅ represents a largehydrophobic amino acid sidechain; R₆ represents alkyl, cycloalkyl, aryl,heterocycle or —(CH₂)_(n)—R₇; R₇ represents aryl, aralkyl, cycloalkyl,alkoxy, alkylthio, —OH or —SH; R₁₁ represents H or lower alkyl; Wrepresents —CN, an epoxyketone, —CH═NR₈,

R₈ represents H, alkyl, alkenyl, alkynyl, —C(X₁)(X₂)X₃, —(CH₂)_(m)—R₉,—(CH₂)_(n)—OH, —(CH₂)_(n)—O-alkenyl, —(CH₂)_(n)—O-alkynyl,—(CH₂)_(n)—O-alkynyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₉, —(CH₂)_(n)—SH,—(CH₂)_(n)—S-alkyl, —(CH₂)_(n)—S-alkenyl, —(CH₂)_(n)—S-alkynyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₉, —C(═O)C(═O)NH₂, —C(═O)C(═O)OR₁₀; R₉represents, independently for each occurrence, a substituted orunsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;R₁₀ represents, independently for each occurrence, hydrogen, or asubstituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle; Y₁ and Y₂ can independently or together beOH, or a group capable of being hydrolyzed to a hydroxyl group,including cyclic derivatives where Y₁ and Y₂ are connected via a ringhaving from 5 to 8 atoms in the ring structure; R₅₀ represents O or S;R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₁₀; R₅₂ represents hydrogen,lower alkyl, amine, —OR₁₀, or a pharmaceutically acceptable salt, or R₅₁and R₅₂ taken together with the phosphorous atom to which they areattached complete a heterocyclic ring having from 5 to 8 atoms in thering structure; X₁ is halogen; X₂ and X₃ each represent H or halogen; mis zero or an integer in the range of 1 to 8; and n is an integer in therange of 1 to
 8. 3. The FAP-activated proteasome inhibitor of claim 2,represented by


4. A pharmaceutical composition, comprising a FAP-activated proteasomeinhibitor of claim 1; and a pharmaceutically acceptable excipient.
 5. Amethod of inhibiting proteasome function in a cell, comprisingcontacting the cell with an effective amount of a FAP-activatedproteasome inhibitor of claim
 1. 6. A method of inhibiting antigenpresentation in a cell, comprising contacting the cell with an effectiveamount of a FAP-activated proteasome inhibitor of claim
 1. 7. A methodof treating cancer, psoriasis, restenosis, or other cell proliferativedisease, comprising administering to a mammal in need thereof atherapeutically effective amount of a FAP-activated proteasome inhibitorof claim
 1. 8. The method of claim 7, further comprisingco-administering to the mammal in need thereof a therapeuticallyeffective amount of a chemotherapeutic agent.
 9. The method of claim 8,wherein the chemotherapeutic agent is docetaxel, paclitaxel, imatinibmesylate, gemcitabine, cis-platin, carboplatin, 5-fluorouracil,pemetrexed, methotrexate, doxorubicin, lenalidomide, dexamethasone, ormonomethyl auristatin.
 10. The method of claim 8, wherein thechemotherapeutic agent is docetaxel, gemcitabine, carboplatin, ordoxorubicin.
 11. The method of claim 8, wherein the chemotherapeuticagent is MG-132, PSI, fellutamide B, bortezomib, CEP-18770, MLN-2238,MLN-9708, epoxomicin, carfilzomib (PR-171), NC-005, YU-101, LU-005,YU-102, NC-001, LU-001, NC-022, PR-957 (LMP7), CPSI (β5), LMP2-sp-ek,BODIPY-NC-001, azido-NC-002, ONX-0912, omuralide, PS-519, marizomib,belactosin A, ¹²⁵I-NIP-L₃VS, NC-005-VS, or MV151.
 12. A method ofinhibiting HIV infection in a mammal, comprising administering to amammal in need thereof a therapeutically effective amount of aFAP-activated proteasome inhibitor of claim
 1. 13. The FAP-activatedproteasome inhibitor of claim 2, wherein the acyl N-terminal blockinggroup is selected from the group consisting of formyl, acetyl, benzoyl,trifluoroacetyl, succinyl and methoxysuccinyl.
 14. The FAP-activatedproteasome inhibitor of claim 2, wherein the acyl N-terminal blockinggroup is represented by the formula —C(═O)—(CH₂)₁₋₁₀—C(═O)—OH.
 15. TheFAP-activated proteasome inhibitor of claim 2, wherein the acylN-terminal blocking group is succinyl.
 16. The FAP-activated proteasomeinhibitor of claim 2, wherein the acyl N-terminal blocking group isselected from the group consisting of aryl(C₁-C₆)acyl, andheteroaryl(C₁-C₆)acyl.
 17. The FAP-activated proteasome inhibitor ofclaim 16, wherein the acyl N-terminal blocking group is anaryl(C₁-C₆)acyl, wherein aryl(C₁-C₆)acyl is a (C₁-C₆)acyl substitutedwith an aryl selected from the group consisting of benzene, naphthalene,phenanthrene, phenol and aniline.
 18. The FAP-activated proteasomeinhibitor of claim 16, wherein the acyl N-terminal blocking group is anheteroaryl(C₁-C₆)acyl, wherein heteroaryl(C₁-C₆)acyl is a (C₁-C₆)acylsubstituted with a heteroaryl selected from the group consisting ofpyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine.
 19. Apharmaceutical composition, comprising a FAP-activated proteasomeinhibitor of claim 2; and a pharmaceutically acceptable excipient.
 20. Amethod of inhibiting proteasome function in a cell, comprisingcontacting the cell with an effective amount of a FAP-activatedproteasome inhibitor of claim
 2. 21. A method of inhibiting antigenpresentation in a cell, comprising contacting the cell with an effectiveamount of a FAP-activated proteasome inhibitor of claim
 2. 22. A methodof treating cancer, psoriasis, restenosis, or other cell proliferativedisease, comprising administering to a mammal in need thereof atherapeutically effective amount of a FAP-activated proteasome inhibitorof claim
 2. 23. The method of claim 22, further comprisingco-administering to the mammal in need thereof a therapeuticallyeffective amount of a chemotherapeutic agent.
 24. The method of claim23, wherein the chemotherapeutic agent is docetaxel, paclitaxel,imatinib mesylate, gemcitabine, cis-platin, carboplatin, 5-fluorouracil,pemetrexed, methotrexate, doxorubicin, lenalidomide, dexamethasone, ormonomethyl auristatin.
 25. The method of claim 23, wherein thechemotherapeutic agent is docetaxel, gemcitabine, carboplatin, ordoxorubicin.
 26. The method of claim 23, wherein the chemotherapeuticagent is MG-132, PSI, fellutamide B, bortezomib, CEP-18770, MLN-2238,MLN-9708, epoxomicin, carfilzomib (PR-171), NC-005, YU-101, LU-005,YU-102, NC-001, LU-001, NC-022, PR-957 (LMP7), CPSI (β5), LMP2-sp-ek,BODIPY-NC-001, azido-NC-002, ONX-0912, omuralide, PS-519, marizomib,belactosin A, ¹²⁵I-NIP-L₃VS, NC-005-VS, or MV151.
 27. A method ofinhibiting HIV infection in a mammal, comprising administering to amammal in need thereof a therapeutically effective amount of aFAP-activated proteasome inhibitor of claim 2.